A biggest challenge in the field of drug discovery is development of drugs that exhibit high medicinal effects with few side effects. Therefore, currently being focused on is pharmacokinetics, in particular, concentration monitoring (therapeutic drug monitoring, TDM). Screening of seeds is performed based on whether or not a prescribed medicine is of an adequate amount and whether or not the medicine reaches a lesion site as indicators, and thereby, drugs to drop out are discovered at an early stage, and this information can be effectively used in early drug discovery development and clinical trials. In particular, in fields such as cancer and autoimmune diseases, drug discovery called molecularly targeted drugs is mainstream these days. It is important that an effective pharmacological effect of a molecularly targeted drug can be judged by a doctor himself or herself by measuring whether or not the drug accumulates at a lesion site and exerts its medicinal effect.
Currently, antibody drugs that use a pathogenic protein as a target antigen are attracting attention as molecularly targeted drugs. Since an antibody is a protein that naturally present in the body, side effects are expected to be low, and it is also possible to administer an antibody at a high concentration to enhance a molecular target effect. Further, due to its nature, an antibody has been said to exhibit extremely high molecular specificity and accumulate in a target lesion. However, there is also a problem of drug prices of antibody drugs, and it is argued that it is important to conduct optimized medical care by proper use of antibody drugs. Therefore, it is also important to set an optimal dosage by measuring localization and concentration of an antibody drug. Further, there is also a great demand for quantification of concentration of an antibody drug in order to proper observe a drug efficacy indicator of an administered drug.
A most common technique for quantification of a protein such as an antibody is ELISA (Enzyme-Linked ImmunoSorbent Assay). This is a technique for easily quantifying molecules to be measured by preparing an antibody with respect to a protein to be measured and further sandwiching with detection antibodies. ELISA is an extremely versatile technology and an automated support environment is also in place. Therefore, ELISA is expected to become a golden standard as a diagnostic technology from now on.
However, for example, ELISA does not directly measure a target to be measured. Therefore, there are many problems such as that an abnormal value may be generated, that it takes time and cost since it is necessary to produce an antibody for each target to be measured, and that it is impossible to simultaneously measure multiple analysis targets. In particular, for an antibody drug, a cross reaction with an endogenous antibody also occurs, and thus an accurate measurement is difficult. Further, in a state in which a neutralizing antibody and an antigen are bound to each other, an antigen recognition site is blocked and it is often not possible to perform measurement using ELISA.
Further, under analysis conditions of ELISA adopted in an animal test phase, due to a problem of interspecific crossing, it is often not directly applicable to large animals and humans. That is, in a drug discovery development stage and in a human clinical trial, it is inevitable to perform comparison under separate measurement conditions. In ELISA of a drug concentration in a lesion tissue, matrix components that inhibit detection are different. Therefore, in order to perform pharmacokinetics based on ELISA, it is necessary to prepare multiple antibodies. This has a very large risk such as enormous cost and dropout in late-stage development.
On the other hand, quantitative and structural analysis of proteins using mass spectrometry has been dramatically expanded in its application range, along with developments of mass spectrometry technologies and data analysis server and software. In particular, an absolute quantification technique using mass spectrometry has increased awareness as a method independent of specific antibodies.
For example, when quantifying a protein without a commercially available antibody, conventionally, it was necessary to purify the protein in a large amount. However, by using mass spectrometry, this step is omitted and thus it becomes dramatically efficient. Even in the field of medicine, conventionally, due to problems such as procedures of doctors such as a method for thin sectioning of a pathological section and a method of preservation, a difference occurs in immunohistochemical staining, and it is often difficult to judge positivity, false positivity, and negativity. In contrast, by quantifying a target pathogenic protein in a pathological section using mass spectrometry, it is possible to judge whether or not the protein is a highly expressed protein. Further, in recent years, a device called a laser microdissection that cuts out one cell is generically used. For example, it is possible to collect only cancer cells and directly observe expression variation analysis of a pathogenic protein in the cells using mass spectrometry. This is a very innovative technological innovation, especially in pathology and in clinical practice, and preparation such as standardization of analytical techniques is desired.
While it is a highly accurate analytical technique, when a protein in a biological sample is detected using mass spectrometry, a protein to be measured is often fragmented by proteolysis. Therefore, it is important to efficiently select a target peptide fragment from various peptide fragments including contaminants.
Patent Document 1 discloses that, in order to detect an antibody in a sample, an F(ab′)2 fragment is produced by using pepsin to decompose a non-immunoglobulin protein and digest an antibody, and thereafter, trypsin digestion is further performed. Further, Patent Document 2 discloses that only a peptide appropriately separated at a liquid chromatography stage prior to mass spectrometry is selected as a quantification target. Non-Patent Document 1 discloses that an anti-peptide antibody is used to concentrate a peptide to be measured.
Further, in recent years, as a method for improving efficiency of protease digestion, a method has attracted attention in which protease digestion is performed in a microenvironment (microreactor) such as a nanoparticle. For example, Non-Patent Document 2 reports that, by using mesoporous silica in which trypsin is immobilized in the pores, it is possible to selectively trypsin digest a protein with a small molecular weight. Non-Patent Document 3 reports an example in which trypsin is immobilized on a nylon membrane and high efficiency of trypsin digestion of a protein is achieved. In all of these methods, a protease is immobilized in pores of a porous body, and the protease on a solid surface and a substrate protein in a liquid phase are caused to react.
Non-Patent Document 4 proposes a high-throughput method for identifying a monoclonal antibody and an endogenous antibody in a sample.